Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.     

Subdomain II of α-Isopropylmalate Synthase Is Essential for Activity: INFERRING A MECHANISM OF FEEDBACK INHIBITION*

The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product l-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.     

Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase.

The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA-specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA-binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site-directed mutations within the highly conserved CHAE sequence of the postulated C-terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2

The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.     

The Role of ACT-Like Subdomain in Bacterial Threonine Dehydratases

In bacteria, threonine dehydratases could convert L-threonine to 2-ketobutyrate. Some threonine dehydratases contain only a catalytic domain, while others contain an N-terminal catalytic domain and a C-terminal regulatory domain composed of one or two ACT-like subdomains. However, the role of the ACT-like subdomain in threonine dehydratases is not clear. Here, nine different bacterial threonine dehydratases were studied. Three of the nine contain no ACT-like subdomain, four of them contain a single ACT-like subdomain, and two of them contain two ACT-like subdomains. The nine genes encoding these threonine dehydratases were individually overexpressed in E. coli BL21(DE3), and the enzymes were purified to homogeneity. Activities of the purified enzymes were analyzed after incubation at different temperatures and different pHs. The results showed that threonine dehydratases with a single ACT-like subdomain are more stable at higher temperatures and a broad range of pH than those without ACT-like subdomain or with two ACT-like subdomains. Furthermore, the specific activity of threonine dehydratases increases with the increase of the number of ACT-like subdomains they contain. The results suggest that the ACT-like subdomain plays an important role in bacterial threonine dehydratases.     

NMR solution structure of the RED subdomain of the Sleeping Beauty transposase

DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi‐domain protein containing the catalytic and the DNA‐binding domains. The DNA‐binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.     

Crystal structure of the C-terminal 10-kDa subdomain of Hsc70

The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions. They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain. The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain. Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 A. Its helical region adopted a helix-loop-helix fold. This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70. Moreover, in the crystalline state, the 10-kDa subdomain formed dimers. The results of gel filtration chromatography further supported the view that this subdomain was self-associated. Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers. These findings suggest that the alpha-helical region of the 10-kDa subdomain dictates the chaperone self-association.     

Critical role of a subdomain of the N-terminus of the V1a vasopressin receptor for binding agonists but not antagonists; functional rescue by the oxytocin receptor N-terminus

A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The V(1a) receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone arginine-vasopressin (AVP). Here we define a short subdomain of the N-terminus of the V(1a)R from Glu(37) to Asn(47) that is an absolute requirement for binding AVP and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The oxytocin receptor (OTR) also binds AVP with high affinity but exhibits a different pharmacological profile to the V(1a)R. Substitution of the N-terminus of the V(1a)R with the corresponding sequence from the OTR generated a chimeric receptor (OTR(N)-V(1a)R). The presence of the OTR N-terminus recovered high affinity agonist binding such that the OTR(N)-V(1a)R possessed almost wild-type V(1a)R pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the V(1a)R N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.     

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis,implicated in developmental processes

PrkC was shown to be a eukaryotic-like (Hanks-type) protein kinase from Bacillus subtilis with a structural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF beta or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane-spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co-transcribed with prkC, is also required for normal biofilm and spore formation.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

Involvement of Both Dockerin Subdomains in Assembly of the Clostridium thermocellum Cellulosome

Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.     

Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity

Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis. Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI. However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity. In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria. Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI. The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains. However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed. The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.     

Inter- and intragenus structural variations in caliciviruses and their functional implications

The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.     

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.     

Crystal structures of the type III effector protein AvrPphF and its chaperone reveal residues required for plant pathogenesis

The avrPphF locus from Pseudomonas syringae pv. phaseolicola, the causative agent of bean halo-blight disease, encodes proteins which either enhance virulence on susceptible hosts or elicit defense responses on hosts carrying the R1 resistance gene. Here we present the crystal structures of the two proteins from the avrPphF operon. The structure of AvrPphF ORF1 is strikingly reminiscent of type III chaperones from bacterial pathogens of animals, indicating structural conservation of these specialized chaperones, despite high sequence divergence. The AvrPphF ORF2 effector adopts a novel "mushroom"-like structure containing "head" and "stalk" subdomains. The head subdomain possesses limited structural homology to the catalytic domain of bacterial ADP-ribosyltransferases (ADP-RTs), though no ADP-RT activity was detected for AvrPphF ORF2 in standard assays. Nonetheless, this structural similarity identified two clusters of conserved surface-exposed residues important for both virulence mediated by AvrPphF ORF2 and recognition of this effector by bean plants expressing the R1 resistance gene.     

Production of protective antibodies against Porphyromonas gingivalis strains by immunization with recombinant gingipain domains

We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain, r-Rgp CAT; hemagglutinin domains, r-Rgp 44 and r-Rgps 15-27) and one fragment of Kgp (catalytic domain, r-Kgp CAT). Enhancement of opsonization and killing by human polymorphonuclear leukocytes were measured in the noninvasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 was the most effective in both strains of P. gingivalis. The present findings lead us to recommend RgpA 44 as a candidate immunogen for vaccines against P. gingivalis.     

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed.Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms.We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner.As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.